Knockout of ovary serine protease Leads to Ovary Deformation and Female Sterility in the Asian Corn Borer, Ostrinia furnacalis

Oogenesis in insects is a carefully orchestrated process, facilitating the formation of female gametes, which is regulated by multiple extrinsic and intrinsic factors, including ovary serine protease (Osp). As a member of the serine protease family, Osp is a homolog of Nudel, a maternally required protease defining embryonic dorsoventral polarity in Drosophila. In this study, we used CRISPR/Cas9-mediated mutagenesis to functionally characterize Osp in the Asian corn borer, Ostrinia furnacalis, a devastating maize pest throughout Asia and Australia. Building on previous knowledge, we hypothesized that knockout of Osp would disrupt embryonic development in O. furnacalis females. To examine this overarching hypothesis, we (1) cloned and characterized Osp from O. furnacalis, (2) designed target sites on exons 1 and 4 to construct a CRISPR/Cas9 mutagenesis system, and (3) documented phenotypic impacts among O. furnacalis Osp mutants. As a result, we (1) examined the temporal-spatial expression profiles of OfOsp, which has an open reading frame of 5648 bp in length and encodes a protein of 1873 amino acids; (2) established O. furnacalis Osp mutants; and (3) documented recessive, female-specific sterility among OfOspF mutants, including absent or deformed oviducts and reduced fertility in female but not male mutants. Overall, the combined results support our initial hypothesis that Osp is required for embryonic development, specifically ovarian maturation, in O. furnacalis females. Given its substantial impacts on female sterility, Osp provides a potential target for the Sterile Insect Technique (SIT) to manage Lepidoptera pests in general and the species complex Ostrinia in particular.


Introduction
The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae), is an important corn pest that significantly threatens crop yields and quality [1].The geographic range of O. furnacalis extends throughout Asia, the Western Pacific, and Australia [2,3].The larvae of O. furnacalis mostly infest the ears and leaves of corn and bore tunnels in corn stems, resulting in crop losses of up to 30% in cases of severe infestation.Damage caused by O. furnacalis can lead to fungal contamination, which further influences the quality of fresh corn [4].In addition to corn, O. furnacalis infects a variety of other hosts including soybeans, wheat, cotton, sorghum, and millet [5,6].Control of O. furnacalis relies on chemical pesticides and some biological control agents, such as parasitic wasps [7] and the insecticidal Bt toxin [8,9].However, overuse of pesticides is associated with the development of resistance and environmental concerns [10,11].Thus, an alternative strategy to control O. furnacalis, the most destructive pest of corn, is urgently needed.
Successful application of the Sterile Insect Technique (SIT) is dependent on the release of sterile insects into the field, which mate with wildtype insects and either produce abnormal offspring or eliminate offspring altogether, thereby controlling the target insect [12,13].
Serine proteases work as proteolytic enzymes that modulate a variety of physiological processes, such as digestion, signal transduction, and defense reactions [25,26], and are also involved in sperm differentiation and function in mammals [27,28].In insects, serine proteases are required for post-mating responses in female D. melanogaster [29].Recent studies in lepidopteran insects have also verified the vital role of serine proteases in male sterility and female reproductivity [30,31].Ovarian serine protease (Osp) is homologous to Nudel in D. melanogaster [32].Osp is expressed by ovarian follicular cells during mid and late vitellogenesis in Bombyx mori [31].Nudel protein is expressed as an extracellular matrix-like molecule that may be anchored in the vitelline membrane [33].Nudel is responsible for specifying embryonic dorsoventral polarity, development of embryogenesis, and oogenesis in D. melanogaster, as well as increasing the structural integrity of the egg [33].Mutations in Nudel have weakened the structural integrity of eggs, hindered development, and ultimately led to embryonic death, resulting in female infertility [34].In B. mori, Osp protein shares 65% of its identity with the homologous Drosophila Nudel protein, and mutagenesis of Osp leads to abnormal oogenesis and female sterility that can be inherited by offspring, indicating the conserved function of Osp protein in lepidopteran insects [31].
In this study, we aimed to functionally characterize the role of O. furnacalis Osp, OfOsp, in ovarian development and reproduction.We hypothesized that OfOsp disrupts embryonic development in O. furnacalis females.To examine this hypothesis, we (1) cloned and characterized OfOsp, (2) knocked out OfOsp in O. furnacalis using the CRISPR/Cas9mediated gene-editing system, and (3) documented the morphological and physiological consequences caused by OfOsp mutagenesis.

Cloning of OfOsp
The coding sequence of OfOsp (XM_028323031) was obtained by checking BLAST B. mori Osp (GenBank: NM_001043703.1)against the O. furnacalis genome [35].As a result, a homologous Osp protein (XP_028178832.1) that shares 70.61% of its identity with B. mori Osp (NP_001037168.1) was identified.OfOsp is 5648 bp in length and encodes 1873 amino acids.Primers were designed to amplify approximately 1500-2500 bp fragments, and the full-length Osp open reading frame (ORF) was assembled from the resulting sequencing data (Table 1).After sequencing, the full length OfOsp ORF was obtained and was used to design sgRNAs.

Primer Name
Primer Sequence (5'-3') Preparation of sgRNA templates In S. litura, Osp exhibits relatively high expression in female pupae and adults, with tissue-specific expression in the ovaries [31].In O. furnacalis, the transcript levels of OfOsp in fourth-instar larvae, fifth-instar larvae, female pupae, and female adults were significantly higher compared to those in the early 12 h old eggs post-laying (p < 0.05).The highest transcript level of OfOsp was observed in adult females, which was about 46-fold higher than that in the egg stage, and this was followed by female pupae (Figure 1A), suggesting that OfOsp plays an important role in the formation of ovaries in pupae and adults.As for the spatial distribution, the transcript level of OfOsp was approximately 23-fold higher in the ovaries of adult females than in adult heads (Figure 1B).These temporal and spatial expression patterns support the potential role of OfOsp in ovarian development during the pupal and adult stages (Figure 1).
suggesting that OfOsp plays an important role in the formation of ovaries in pupae and adults.As for the spatial distribution, the transcript level of OfOsp was approximately 23fold higher in the ovaries of adult females than in adult heads (Figure 1B).These temporal and spatial expression patterns support the potential role of OfOsp in ovarian development during the pupal and adult stages (Figure 1).The temporal distribution of OfOsp in eggs at 12h, 24h, 48h and 72h, larvae at instars 1 to 5, female and male pupae, and female and male adults was examined.(B) The spatial expression analysis from tissues of heads, thoraxes (with no legs and wings), legs, forewings, hindwings, abdominal epidermis, fat bodies, ovaries, and external genitalia was examined.One-way ANOVA in SPSS statistics 22 was used for data analysis."ns" stands for "not significant" (p > 0.05).The asterisks (* and ***) indicate significant differences (p < 0.05 and p < 0.001).

Mutagenesis of OfOsp using CRISPR/Cas9
OfOsp contains 22 exons and 21 introns (Figure 2A).CRISPR/Cas9-mediated mutagenesis was used to investigate the functionality of OfOsp in O. furnacalis.Two sgRNAs targeting two different positions on exons 1 and 4 of OfOsp were designed, and the positions of the sgRNAs on these exons are illustrated in Figure 2A.We injected 347 eggs with Cas9 protein and the two sgRNAs and obtained a 61.64% hatching rate and a 28.42% mutation rate in the treated eggs.All insertions or deletions resulted in premature stop codons, leading to truncation or loss of function of the OfOsp protein.The female mutants had a 989 bp fragment missing from exons 1 to 4, whereas male mutants showed a deletion of 976 bp from exons 1 to 4, producing an N-terminal truncated OfOsp protein (Figure 2B).DNA from molted OfOsp mutants and wildtype individuals was extracted and sequenced, demonstrating that OfOsp was successfully mutated (Figure 2C).Using primers flanking the two target sites at exons 1 and 4 in mutant and WT adults, the resultant banding pattern further indicated that OfOsp was successfully mutated (Figure 2D).The temporal distribution of OfOsp in eggs at 12 h, 24 h, 48 h and 72 h, larvae at instars 1 to 5, female and male pupae, and female and male adults was examined.(B) The spatial expression analysis from tissues of heads, thoraxes (with no legs and wings), legs, forewings, hindwings, abdominal epidermis, fat bodies, ovaries, and external genitalia was examined.One-way ANOVA in SPSS statistics 22 was used for data analysis."ns" stands for "not significant" (p > 0.05).The asterisks (* and ***) indicate significant differences (p < 0.05 and p < 0.001).

Mutagenesis of OfOsp Using CRISPR/Cas9
OfOsp contains 22 exons and 21 introns (Figure 2A).CRISPR/Cas9-mediated mutagenesis was used to investigate the functionality of OfOsp in O. furnacalis.Two sgRNAs targeting two different positions on exons 1 and 4 of OfOsp were designed, and the positions of the sgRNAs on these exons are illustrated in Figure 2A.We injected 347 eggs with Cas9 protein and the two sgRNAs and obtained a 61.64% hatching rate and a 28.42% mutation rate in the treated eggs.All insertions or deletions resulted in premature stop codons, leading to truncation or loss of function of the OfOsp protein.The female mutants had a 989 bp fragment missing from exons 1 to 4, whereas male mutants showed a deletion of 976 bp from exons 1 to 4, producing an N-terminal truncated OfOsp protein (Figure 2B).DNA from molted OfOsp mutants and wildtype individuals was extracted and sequenced, demonstrating that OfOsp was successfully mutated (Figure 2C).Using primers flanking the two target sites at exons 1 and 4 in mutant and WT adults, the resultant banding pattern further indicated that OfOsp was successfully mutated (Figure 2D).

Phenotypic and Physiological Impacts Experienced by OfOsp Mutants 2.3.1. Phenotypic Impacts on Internal Genitalia
The genitalia of wildtype and OfOsp mutant O. furnacalis adults were dissected to observe the phenotypic changes associated with mutagenesis.The ovaries and testes of mutant O. furnacalis adults, together with those from wildtype groups, were dissected on the third day post-eclosion (Figure 3).Wildtype females possessed ovaries supplemented with four pairs of oviducts that were localized on two sides (Figure 3A).As expected, female OfOsp F mutants had severe malformations of the ovaries, having one oviduct missing on one side, leaving seven oviducts remaining (Figure 3B), with a closeup view of the oviducts shown in Figure 3D.Additionally, we observed only two pairs of oviducts that were shortened relative to the wildtype females and generated fewer or smaller eggs within the oviducts of OfOsp F compared to the wildtypes (Figure 3C).The ovaries of OfOsp F mutants also showed fused and shortened oviducts that either produced fewer eggs or failed to develop normally (Figure 3C).Male OfOsp mutants presented normal phenotypes with no phenotypic changes observed compared to the wildtypes (Figure 3E,F).

Phenotypic Impacts on Internal Genitalia
The genitalia of wildtype and OfOsp mutant O. furnacalis adults were dissected to observe the phenotypic changes associated with mutagenesis.The ovaries and testes of mutant O. furnacalis adults, together with those from wildtype groups, were dissected on the third day post-eclosion (Figure 3).Wildtype females possessed ovaries supplemented with four pairs of oviducts that were localized on two sides (Figure 3A).As expected, female OfOsp F mutants had severe malformations of the ovaries, having one oviduct missing on one side, leaving seven oviducts remaining (Figure 3B), with a closeup view of the oviducts shown in Figure 3D.Additionally, we observed only two pairs of oviducts that were shortened relative to the wildtype females and generated fewer or smaller eggs within the oviducts of OfOsp F compared to the wildtypes (Figure 3C).The ovaries of OfOsp F mutants also showed fused and shortened oviducts that either produced fewer eggs or failed to develop normally (Figure 3C).Male OfOsp mutants presented normal phenotypes with no phenotypic changes observed compared to the wildtypes (Figure 3E,F).

Effects of OfOsp Mutagenesis on Fertility and Fecundity
Ovary development is important for the fertility and fecundity of female adults.To investigate if OfOsp mutagenesis leads to adult sterility, the fecundity and hatching rates of both mutant and wildtype O. furnacalis were recorded continuously for ten days after pairing individuals with wildtype individuals of the opposite sex.In wildtype pairings, females laid an average of 483 ± 34 eggs within the ten-day period.OfOsp F mutants laid an average of 113 ± 16 eggs within the ten-day period, which is significantly decreased compared to wildtype females.Wildtype females paired with OfOsp mutant males laid 340 ± 65 eggs within the ten-day period, which was not significantly different from pure wildtype pairings (Figure 4A).The hatching rate of eggs laid by OfOsp female and male mutants showed no significant differences in comparison to the wildtypes (Figure 4B).

Pleiotropic Effects of OfOsp Mutagenesis
Given that Vitellogenin (Vg) and Vitellogenin receptor (VgR) are associated with the process of vitellogenesis in insects [36], we examined the relative transcript levels of Vg and VgR in the ovaries of O. furnacalis after OfOsp mutagenesis.The relative transcript level of pipe was also examined in OfOsp mutants, where pipe was significantly upregulated in OfOsp F mutants by 3.08-fold compared to the wildtypes (Figure 5A).The results showed that Vg was upregulated by 2.72-fold when normalized to the wildtypes after OfOsp mutagenesis (Figure 5B).The relative transcript level of VgR was not changed (Figure 5C).In Drosophila, Pipe together with Nudel are required for embryonic dorsal-ventral polarity [37].We also further verified the transcript levels of OfOsp in the ovaries of OfOsp F mutants, which were significantly decreased compared to the wildtypes (Figure 5D).

Pleiotropic Effects of OfOsp Mutagenesis
Given that Vitellogenin (Vg) and Vitellogenin receptor (VgR) are associated with the process of vitellogenesis in insects [36], we examined the relative transcript levels of Vg and VgR in the ovaries of O. furnacalis after OfOsp mutagenesis.The relative transcript level of pipe was also examined in OfOsp mutants, where pipe was significantly upregulated in OfOsp F mutants by 3.08-fold compared to the wildtypes (Figure 5A).The results showed that Vg was upregulated by 2.72-fold when normalized to the wildtypes after OfOsp mutagenesis (Figure 5B).The relative transcript level of VgR was not changed (Figure 5C).In Drosophila, Pipe together with Nudel are required for embryonic dorsal-ventral polarity [37].We also further verified the transcript levels of OfOsp in the ovaries of OfOsp F mutants, which were significantly decreased compared to the wildtypes (Figure 5D).
that Vg was upregulated by 2.72-fold when normalized to the wildtypes after OfOsp tagenesis (Figure 5B).The relative transcript level of VgR was not changed (Figure 5C Drosophila, Pipe together with Nudel are required for embryonic dorsal-ventral pola [37].We also further verified the transcript levels of OfOsp in the ovaries of OfOsp F tants, which were significantly decreased compared to the wildtypes (Figure 5D).

Identification and Characterization of OfOsp
Nudel is expressed during oogenesis and is essential for egg development after fertilization [33].In lepidopterans, Osp was first analyzed as a homolog to Drosophila Nudel in B. mori and S. litura [31].Mutagenesis of OSP in these two species causes abnormal development in the ovaries and female sterility [31] (Figure S1).Using the published O. furnacalis genome [35], we identified a homologous Osp protein that shares 70.61% of its identity with the B. mori Osp, and phylogenetic analysis supports that Osp is highly conserved among lepidopteran insects (Figure S1).
Osp is highly expressed in the ovaries of female pupae and adults in lepidopteran insects [31].In our study, temporal, and spatial expression patterns of OfOsp were analyzed to show its increased expression in the ovarian tissues of female adults, supporting its essential role in germ line formation and oogenesis during embryonic development (Figure 1A).Temporal expression verified the expression level of OfOsp in different developmental stages, showing that OfOsp is highly expressed in the fourth-and fifth-instar larvae, female pupae, and female adults of O. furnacalis (Figure 1B).

Mutagenesis of OfOsp Using CRISPR/Cas9 Gene Editing System
The specificity and flexibility of the CRISPR/Cas9 system allows precise editing of genes that would benefit strategies for pest management.In the mosquito Anopheles gambiae, using CRISPR/Cas9 to function as a gene drive system, three putative female fertilityrelated genes were identified and targeted to successfully suppress mosquito populations that transmit malaria [32].S. frugiperda was disrupted through embryonic microinjection of Cas9 protein and in vitro-transcribed sgRNAs targeting doublesex, leading to impaired ovaries and testes in adults and an inheritable reduction in fertility and fecundity [38].Our results show that mutagenesis of OfOsp results in missing and malformed oviducts, with oviducts in some cases fusing together, disabling their ability to produce normal eggs (Figure 3).In B. mori, knockout of Osp leads to irregular oviducts and shorter and shrunken oviducts in mutants [31].Our results have further verified the functional role of Osp in ovarian development and oogenesis in lepidopterans.

Phenotypic Impacts among OfOsp Mutants
Regulating and disrupting the secretion of proteins from insect reproductive glands as a means of pest control has become a new strategy in the field of insect infertility technology.Gligorov and colleagues discovered that the absence of Abd-B in D. melanogaster resulted in disruption of the ability of females to re-mate with males and to lay eggs when mating with mutated male individuals [39].Similarly, knockout of Abd-A and Ubx leads to defects in wing formation and reduced reproductivity in O. furnacalis [40].Other potential targets for SIT to control O. furnacalis are Masc and doublesex, the mutagenesis of which leads to reduced fecundity and fertility [24].Our results have identified an additional target to control O. furnacalis via the release of mutant males that carry a mutated OfOsp to mate with females in the wild.In this study, we observed the development of abnormal ovaries in female OfOsp mutants and normal testes in males (Figure 3).As a result, the fertility of female, not male, mutants is significantly decreased after OfOsp knockdown (Figure 4), indicating the functional role of OfOsp in ovarian development and oogenesis.This result is inconsistent with the results for B. mori and S. litura, in which knockdown of Osp significantly reduces fertility [31].
Insect oogenesis requires expression of a variety of genes.Pipe is required in the somatic tissue of Drosophila egg chambers and defines embryonic dorsal-ventral polarity [37].We first examined the relative expression of OfOsp in the dissected ovaries in OfOsp mutants and found that knockout of OfOsp significantly decreases the expression of OfOsp (Figure 5D).Since Osp and Pipe are products of dorsal-group genes that play key roles in spatially regulating the protease cascade involved in controlling embryonic patterning and polarity in Drosophila [41], we examined the relative transcript levels of pipe and found that they were upregulated in OfOsp F mutants compared to the wildtypes (Figure 5A).Previous studies have reported that the Pipe gene is highly expressed in the ovaries of Drosophila [37].Our results have further indicated its complementary role in oogenesis alongside Osp.
RNAi targeting Vg and VgR in Diaphorina citri Kuwayama leads to disturbed formation of eggs and suppresses oviposition by adults [42].We examined the relative transcript levels of Vg and VgR in the dissected ovaries of OfOsp mutants.The transcript level of Vg was upregulated 3.04-fold in comparison to the wildtypes (Figure 5), indicating its role in ovary development as complementary to OfOsp.Our results are in accordance with the known role of Vg as being responsible for the formation of yolk proteins in insect eggs [43].As Vg transcripts accumulated in the ovaries of Culex quinquefasciatus [44], the altered upregulation of Vg in our study has further verified its essential role in the ovarian development.

Insect Rearing and Sexing
The O. furnacalis strain used in this study was collected from corn in Qipanshan County, Shenyang City, Liaoning Province, China (123.471097,41.68383) and was kept in laboratory conditions at a temperature of 25 • C with a relative humidity of 70%, light intensity of 1000 lx, and a photoperiod of 16:8 h L:D.O. furnacalis larvae were fed with a standard lepidopteran artificial diet made with yeast extract, wheat bran, vitamin C, sucrose, and agar, as described previously, every two days [38].Pupae were sexed for pairing post-eclosion, and adults were kept in rectangular-shaped metal rearing cages supplemented with cotton balls, which were soaked with 10% honey water to allow for mating and egg laying.

Cloning of OfOsp
To obtain the coding sequence of OfOsp (XM_028323031), we performed a BLAST search using B. mori Nudel (GenBank: NM_001043703.1)against the O. furnacalis genome in NCBI.Total RNA from a fifth-instar O. furnacalis larva was extracted using TRIeasy reagent (Yeasen, China) to amplify the full length of OfOsp.The extracted RNA was then reverse transcribed into cDNA using the PrimeScript TM Reverse Transcription Kit (Takara, Japan) using 1µg of total RNA as a template.The full-length sequence of OfOsp was 5648 bp in length, encoding 1873 amino acids.Primers were designed to amplify 1500-2000 bp fragments of Osp ORF and the full-length sequence was assembled from the resulting sequencing data.After sequencing (Genewiz biotech company, Suzhou, China), the fulllength sequence of OfOsp was constructed and verified before designing sgRNAs.All primers were designed using primer3 (https://primer3.ut.ee (accessed on 1 May 2000)).

Microinjection of sgRNAs and Detection of OfOsp Mutagenesis
To collect eggs for microinjection, the newly emerged O. furnacalis adults were sexed and paired in a plastic bag.Eggs were collected within 1 h of being laid for microinjection under a microscope (Olympus ZSX16, Tokyo, Japan).The microinjection system used in this study was FemtoJet-4i (Eppendorf, Hamburg, Germany) under a microscope (Olympus ZSX16, Tokyo, Japan).Borosilicate Glass Capillaries, with an inner diameter of 0.58 mm, an outer diameter of 1.0 mm, and a length of 10 cm (World Precision Instruments, Sarasota, FL, USA), were used as the needle to deliver a mixture of 300 ng/µL Cas9 protein and 300 ng/µL sgRNA.After injection, eggs were incubated at 28 • C for about 2 days until hatching and later transferred to sterilized containers supplemented with an artificial diet containing yeast extract, wheat bran, vitamin C, sucrose, and agar [38].All insects were kept under the same rearing condition as described above.
To verify Osp knockout in O. furnacalis, pupal shells were collected for sequence analysis.Specifically, genomic DNA was extracted using the phenol -chloroform method and precipitated with sodium acetate isopropanol in conjunction with proteinase K (Ther-moFisher Scientific, Waltham, MA, USA).Primers targeting the flanking regions of the two target sites were designed to detect gene knockout, and amplification was performed using Hieff Canace Gold High-Fidelity DNA Polymerase (Yeasen, China).Reaction conditions were as follows: pre-denaturation at 98 • C for 3 min; 35 cycles of denaturation at 98 • C for 10 s each, annealing at 55 • C for 20 s, and extension at 72 • C for 30 s; and final extension at 72 • C for 30 s. PCR products were then ligated into a pJET1.2vector (ThermoFisher Scientific, Waltham, MA, USA) and sent for sequencing by the Genewiz Biotech Company (Suzhou, China).

Phenotypic Observation after Mutagenesis
The phenotypic impact of OfOsp knockout on the morphology of virgin adults was observed through dissection using a micro-imaging system (KEYENCE VHX7000, Osaka, Japan).The number of injected eggs, hatching rate of eggs, pupation rate, mutation rate, and sex ratio of eclosed adults were also recorded and are summarized in Table 2. To investigate whether mutagenesis of Osp interferes with the fecundity and fertility of O. furnacalis, two-day old virgin OfOsp mutant adults were collected and paired with the opposite-sex wildtype adults.Specifically, wildtype females were paired with wildtype males, female OfOsp mutants were paired with wildtype males, and male OfOsp mutants were paired with wildtype females.Each O. furnacalis pair was placed in a transparent PVC bag to allow for mating and oviposition.During this period, eggs laid by each pair were observed and recorded on a daily basis for a 10-day assay period.The hatching rate was recorded for each pair as well.Experiments were performed in three replicates with 5-20 pairs per treatment.

Real-Time Quantitative PCR (RT-qPCR)
To study the expression levels of OfOsp across different developmental stages and body parts of O. furnacalis, total RNA was extracted from eggs, larvae at instars 1 to 5, female and male pupae, and female and male adults using Trizol reagent (Invitrogen, USA).For spatial expression analysis, tissue was collected from heads, thoraxes (with no legs and wings), legs, forewings, hindwings, abdominal epidermis, fat bodies, ovaries, and external genitalia, and total RNA was extracted.A PrimeScript TM Reverse Transcription Kit (Takara, Japan) was used to synthesize cDNA from 2 µg of total RNA as the template.Quantitative real-time PCR using OfOsp gene-specific primers (Table 1) was performed to examine the spatiotemporal expression patterns of OfOsp, and OfRPS3 (GenBank: EU275206.2),a suitable reference gene for O. furnacalis, was used as the internal control [47].The reaction conditions included pre-denaturation at 95 • C for 30 s, followed by 40 cycles of denaturation at 95 • C for 5 s each, annealing at 58 • C for 30 s, and extension at 72 • C for 30 s.
To investigate if OfOsp mutagenesis interferes with the transcript levels of other genes, including Pipe (GenBank: XM_028307755.1),Vg (GenBank: QIH04838.1),and VgR (GenBank: QIG55527.1),total RNA was extracted from wildtype O. furnacalis and mutant individuals using Trizol reagent (Invitrogen, Carlsbad, CA, USA).According to the manufacturer's instructions, 2 µg of total RNA was used as the template, and cDNA was synthesized using a PrimeScript TM Reverse Transcription Kit (Takara, Japan).The relative gene expression was calculated following the 2 -∆∆Cq method [48].

Statistical Analysis
Statistical analysis of all data was performed using IBM SPSS statistics 22 using a two-tailed t-test.Data were represented as the means ± SEM in all cases.p < 0.05 was considered a significant difference between treatments and controls.

Conclusions
In this study, the functionality of OfOsp was characterized using CRISPR/Cas9mediated mutagenesis.Our results support our hypothesis that OfOsp is vital for ovarian development and oogenesis in O. furnacalis.Given its role in reproduction and the femalespecific nature of its mutagenesis, OfOsp represents a potential molecular target for future SIT-based control of this global maize pest.

Figure 1 .
Figure 1.The temporal and spatial distributions of OfOsp.Data are shown as the mean ± SEM (n = 3).(A)The temporal distribution of OfOsp in eggs at 12h, 24h, 48h and 72h, larvae at instars 1 to 5, female and male pupae, and female and male adults was examined.(B) The spatial expression analysis from tissues of heads, thoraxes (with no legs and wings), legs, forewings, hindwings, abdominal epidermis, fat bodies, ovaries, and external genitalia was examined.One-way ANOVA in SPSS statistics 22 was used for data analysis."ns" stands for "not significant" (p > 0.05).The asterisks (* and ***) indicate significant differences (p < 0.05 and p < 0.001).

Figure 1 .
Figure 1.The temporal and spatial distributions of OfOsp.Data are shown as the mean ± SEM (n = 3).(A)The temporal distribution of OfOsp in eggs at 12 h, 24 h, 48 h and 72 h, larvae at instars 1 to 5, female and male pupae, and female and male adults was examined.(B) The spatial expression analysis from tissues of heads, thoraxes (with no legs and wings), legs, forewings, hindwings, abdominal epidermis, fat bodies, ovaries, and external genitalia was examined.One-way ANOVA in SPSS statistics 22 was used for data analysis."ns" stands for "not significant" (p > 0.05).The asterisks (* and ***) indicate significant differences (p < 0.05 and p < 0.001).

Figure 2 .
Figure 2. Design of sgRNAs and mutagenesis of OfOsp using CRISPR/Cas9 system.(A) The design of sgRNAs targeting exons 1 and 4 of OfOsp.(B) The number of base pairs deleted in the edited sequences (indicated in parentheses).PAM sequences are highlighted in red."Δ989 bp" refers to the number of base pairs deleted after gene knockout.(C) Target sites of OfOsp using CRISPR/Cas9 system in male and female O. furnacalis (labeled with red arrows).(D) Fragments corresponding to Osp mutants and WT in genomic DNA amplified using PCR and resolved with agarose gel electrophoresis.

Figure 2 .
Figure 2. Design of sgRNAs and mutagenesis of OfOsp using CRISPR/Cas9 system.(A) The design of sgRNAs targeting exons 1 and 4 of OfOsp.(B) The number of base pairs deleted in the edited sequences (indicated in parentheses).PAM sequences are highlighted in red."∆989 bp" refers to the number of base pairs deleted after gene knockout.(C) Target sites of OfOsp using CRISPR/Cas9 system in male and female O. furnacalis (labeled with red arrows).(D) Fragments corresponding to Osp mutants and WT in genomic DNA amplified using PCR and resolved with agarose gel electrophoresis.

14 Figure 4 .
Figure 4. Physiological impacts of OfOsp mutagenesis.(A) Fecundity of adult mutants of OfOsp paired with opposite gender of wildtype are shown.(B) The hatching rates of eggs laid by OfOsp female and OfOsp male comparing with wildtypes.Data are shown as the mean ± SEM (n = 5-20), and one-way ANOVA in SPSS statistics 22 was used for data analysis."ns" stands for "not significant" (p > 0.05).The asterisks (***) indicate significant differences (p < 0.0001).The orange circles indicate the value of each replicate recorded for fecundity and hatching rate.

Figure 4 .
Figure 4. Physiological impacts of OfOsp mutagenesis.(A) Fecundity of adult mutants of OfOsp paired with opposite gender of wildtype are shown.(B) The hatching rates of eggs laid by OfOsp female and OfOsp male comparing with wildtypes.Data are shown as the mean ± SEM (n = 5-20), and one-way ANOVA in SPSS statistics 22 was used for data analysis."ns" stands for "not significant" (p > 0.05).The asterisks (***) indicate significant differences (p < 0.0001).The orange circles indicate the value of each replicate recorded for fecundity and hatching rate.

Figure 5 .
Figure 5. Relative expressions of Pipe, Vg, VgR, and OfOsp after OfOsp mutagenesis.The rel transcript levels of Pipe (A), Vg (B) and VgR (C), and OfOsp (D) were examined in ovaries of O female mutants comparing with that in the female ovaries of wildtype .Data are shown as the m ± SEM (n = 4-5), and one-way ANOVA in SPSS statistics 22 was used for data analysis.The or circles indicate values of relative expression of Pipe (A), Vg (B) and VgR (C), and OfOsp (D) nor ized to OfRPS3, the internal reference gene calculated by 2 -ΔΔCq method."ns" stands for

Figure 5 .
Figure 5. Relative expressions of Pipe, Vg, VgR, and OfOsp after OfOsp mutagenesis.The relative transcript levels of Pipe (A), Vg (B) and VgR (C), and OfOsp (D) were examined in ovaries of OfOsp female mutants comparing with that in the female ovaries of wildtype .Data are shown as the mean ± SEM (n = 4-5), and one-way ANOVA in SPSS statistics 22 was used for data analysis.The orange circles indicate values of relative expression of Pipe (A), Vg (B) and VgR (C), and OfOsp (D) normalized to OfRPS3, the internal reference gene calculated by 2 -∆∆Cq method."ns" stands for "not significant" (p > 0.05).The asterisks (*, ** and ***) indicate significant differences (p < 0.05, p < 0.01 and p < 0.001) between mutants and wildtypes. .

Table 1 .
Primers used in this study.

Table 2 .
Statistics of Osp knockout in O. furnacalis.